Use of buckysome or carbon nanotube for drug delivery

ABSTRACT

Compositions and methods for administering a therapeutic agent to a mammal are disclosed. The compositions comprise either (i) vesicles comprising an amphiphilic substituted fullerene, wherein the therapeutic agent is present in the vesicle interior or between layers of the vesicle wall, (ii) a substituted fullerene, comprising a fullerene core and a functional moiety, wherein the therapeutic agent is associated with the substituted fullerene, or (iii) carbon nanotubes, wherein the therapeutic agent is covalently bonded to the carbon nanotubes.

This application claims priority from copending provisional application60/356,856, filed Feb. 14, 2002, the disclosure of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods foradministering a therapeutic agent to a mammal. More particularly, itrelates to vesicles, wherein the vesicle wall comprises substitutedfullerenes and the vesicle comprises the therapeutic agent; derivatizedcarbon nanotubes, wherein the carbon nanotubes are derivatized with thetherapeutic agent; and methods for administering the vesicles orderivatized carbon nanotubes to a mammal.

2. Description of Related Art

In recent years, a variety of approaches have been studied and used fordrug delivery, DNA transfection, and other medical and biologicalapplications. One such set of approaches involves vesicles or liposomes(the two terms will be used interchangeably herein).

Mishra et al., Drug Deliv. (2000) 7(3):155-159 teaches the loading oferythrocyte ghosts with doxorubicin HCl. So-called reverse biomembranevesicles were formed by budding of membrane into the ghost interiors(endocytosis) leading to accumulation of small vesicles within eachparent ghost. The amount of doxorubicin entrapped in reverse biomembranevesicles was 0.75 mg/ml of packed vesicles. The in vitro release profileshowed 52.86% of drug release in 16 hr.

Guo et al., Drug Deliv. (2000) 7(2):113-116 teaches the preparation offlexible lecithin vesicles containing insulin and assessed the effect ofthese vesicles on the transdermal delivery of insulin. When vesicleswere applied onto mice abdominal skin, blood glucose dropped by greaterthan 50% within 18 hr.

Freund, Drug Deliv. (2001) 8(4):239-244 teaches the encapsulation oftherapeutic molecules in a noncationic multilamellar vector comprisingphosphatidylcholine, cholesterol, and polyoxyethylene alcohol. Suchvectors with entrapped drugs were prepared by shearing a phospholipidiclyotropic lamellar phase.

However, a need remains in the art for vesicles which possess propertiessuitable for drug delivery, namely low toxicity of the amphiphiles fromwhich the vesicles are formed and ready vesicle formation anddisaggregation, among others. Such properties are also of interestregarding non-vesicle-based drug delivery systems, as well.

Fullerenes, of which the best known example is C₆₀, were first reportedby Kroto et al., Nature (1985) 318:162. Since then, the readyderivatization of fullerenes has allowed a wide variety of derivatizedfullerenes to be prepared and their properties explored.

Amphiphilic derivatized fullerenes have been reported by Hirsch et al.,Angew. Chem. Int. Ed. (2000) 39(10):1845-1848. The derivatizedfullerenes of Hirsch comprised one dendrimeric group comprising 18carboxylic acid moieties and five hydrophobic moieties each comprising apair of lipophilic C₁₂ hydrocarbon chains. Freeze-fracture electronmicrography of aqueous solutions of the amphiphilic derivatizedfullerenes revealed that the amphiphilic derivatized fullerenes formedbilayer vesicles (by which is meant, a vesicle defined by a membranecomprising an external layer of amphiphilic derivatized fullerenemolecules substantially all oriented with their hydrophilic groups tothe exterior of the vesicle, and an internal layer of amphiphilicderivatized fullerene molecules substantially all oriented with theirhydrophilic groups to the interior of the vesicle, wherein thehydrophobic groups of the molecules of the external layer are in closeproximity to the hydrophobic groups of the molecules of the internallayer) with diameters from about 100 nm to about 400 nm.

Braun et al., Eur. J. Org. Chem. (2000) 1173-1181, teaches the synthesisof biotinated lipofullerenes.

Carbon nanotubes and methods for their derivatization are known.Holzinger et al., Angew. Chem. Int. Ed. (2001) 40(21):4002-4005 reportthe cycloaddition of nitrenes, the addition of nucleophilic carbenes,and the addition of radicals, to the sidewalls of carbon nanotubes.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a vesicle having aninterior, an exterior, and a wall, wherein the wall comprises one ormore layers, wherein each layer comprises a substituted fullerene havingstructure I:(B)_(b)—C_(n)-(A)_(a)   (I)

wherein C_(n) is a fullerene moiety comprising n carbon atoms, wherein nis an integer and 60≦n≦240;

B is an organic moiety comprising from 1 to about 40 polar headgroupmoieties;

b is an integer and 1≦b≦5;

each B is covalently bonded to the C_(n) through 1 or 2 carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds;

A is an organic moiety comprising a terminus proximal to the C_(n) andone or more termini distal to the C_(n), wherein the termini distal tothe C_(n) each comprise —C_(x)H_(y), wherein x is an integer and 8≦x≦24,and y is an integer and 1≦y≦2x+1;

a is an integer, 1≦a≦5;

2≦b+a≦6; and

each A is covalently bonded to the C_(n) through 1 or 2 carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds,

wherein the vesicle wall comprises at least about 50 mol % thesubstituted fullerene, and the interior of the vesicle, a portion of thewall between two layers, or both comprise a therapeutic agent.

In another embodiment, the present invention relates to a method ofadministering a therapeutic agent to a mammal, comprising:

(i) administering a solution comprising a pharmaceutically effectiveamount of the therapeutic agent, wherein the therapeutic agent ispresent in the interior of a vesicle, a portion of the vesicle wallbetween two layers, or both to the mammal, wherein the vesicle is asdefined above.

In yet another embodiment, the present invention relates to a method ofreversibly forming a vesicle comprising a therapeutic agent in theinterior thereof, between two layers of the wall thereof, or both,comprising:

dissolving in an aqueous solvent a substituted fullerene having thestructure I, as described above, and the therapeutic agent,

wherein the pH of the solvent is sufficiently low to form a vesicle fromthe substituted fullerene.

In still another embodiment, the present invention relates to aderivatized carbon nanotube, comprising:

a carbon nanotube, and

at least one therapeutic agent,

wherein each therapeutic agent is covalently attached to the carbonnanotube.

In yet a further embodiment, the present invention relates to a methodof delivering a therapeutic agent to a tissue of a mammal, comprising

(i) administering to the mammal a derivatized carbon nanotube,comprising a carbon nanotube and at least one therapeutic agent, whereineach therapeutic agent is covalently attached to the carbon nanotube,and

(ii) administering to the mammal an adjuvant which promotes disruptionof the covalent bond between the carbon nanotube and the at least onetherapeutic agent when the derivatized carbon nanotube is in proximityto the tissue, thereby delivering the at least one therapeutic agent tothe tissue.

The present invention allows for the convenient preparation ofcompositions that can readily deliver a therapeutic agent to a specifictissue. The ability to target the therapeutic agent to a specific tissueallows the use of smaller doses of the therapeutic agent and may reducesystemic side effects of the therapeutic agent. Further, the substitutedfullerenes and the carbon nanotubes used in the various embodiments ofthe present invention are readily cleared from the body after deliveringthe therapeutic agent.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a particular substituted fullerene (which may be referredto as an “amphifullerene”) of the present invention. The use of gray torepresent three >C(C(═O)O(CH₂)₁₁CH₃)₂ moieties indicates these threemoieties are bonded to the fullerene core at regions of the fullerenewhich are not directly visible to the putative observer in thisorientation.

FIG. 2 shows a cryogenic transmission electron microscopy (“cryo-TEM”)image of a vesicle comprising the amphifullerene represented by FIG. 1.The vesicle has a diameter of about 80 nm and a thickness of the bilayerof about 7 nm. The dark regions in the bilayer represent the C₆₀-core ofthe amphifullerene.

FIG. 3 shows the pressure as a function of a pH for a titration isothermof a monolayer formed from the amphifullerene represented by FIG. 1.

FIG. 4 shows the UV/V is spectrum of the Texas Red® derivative of theamphifullerene represented by FIG. 1. The Texas Red® derivative isreferred to as compound 2 in Scheme 1, Example 3.

FIG. 5 shows the UV/V is spectrum of a partially labeled dendrofullerene(compound 2) with 2.0% fluorophore.

FIG. 6 shows a particular substituted fullerene comprising a functionalgroup, according to the present invention. The use of gray follows thatof FIG. 1. The functional group is a biotin-containing moiety andincludes a linker moiety.

FIG. 7 shows the scheme for synthesis of an amphifullerene labeled withthe fluorescence marker Texas Red®. The use of gray follows that of FIG.1 and FIG. 6.

FIG. 8 shows one embodiment of a method of delivering a therapeuticcompound by the use of a carbon nanotube.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one embodiment, the present invention relates to a vesicle having aninterior, an exterior, and a wall, wherein the wall comprises one ormore layers, wherein each layer comprises a substituted fullerene havingstructure I:(B)_(b)—C_(n)-(A)_(a)   (I)

wherein C_(n) is a fullerene moiety comprising n carbon atoms, wherein nis an integer and60≦n≦240;

B is an organic moiety comprising from 1 to about 40 polar headgroupmoieties;

b is an integer and 1≦b≦5;

each B is covalently bonded to the C_(n) through 1 or 2 carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds;

A is an organic moiety comprising a terminus proximal to the C_(n) andone or more termini distal to the C_(n), wherein the termini distal tothe C_(n) each comprise —C_(x)H_(y), wherein x is an integer and 8≦x≦24,and y is an integer and 1≦y≦2x+1;

a is an integer, 1≦a≦5;

2≦b+a≦6; and

each A is covalently bonded to the C_(n) through 1 or 2 carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds,

wherein the vesicle wall comprises at least about 50 mol % thesubstituted fullerene, and wherein the interior of the vesicle, aportion of the wall between two layers, or both comprise a therapeuticagent.

A “vesicle,” as the term is used herein, is a collection of amphiphilicmolecules, by which is meant, molecules which include both (a)hydrophilic (“water-loving”) regions, typically charged or polarmoieties, such as moieties comprising polar headgroups, among othersknown to one of ordinary skill in the art, and (b) hydrophobic(“water-hating”) regions, typically apolar moieties, such as hydrocarbonchains, among others known to one of ordinary skill in the art. Inaqueous solution, the vesicle is formed when the amphiphilic moleculesform a wall, i.e., a closed three-dimensional surface. The wall definesan interior of the vesicle and an exterior of the vesicle. Typically,the exterior surface of the wall is formed by amphiphilic moleculesoriented such that their hydrophilic regions are in contact with water,the solvent in the aqueous solution. The interior surface of the wallmay be formed by amphiphilic molecules oriented such that theirhydrophilic regions are in contact with water present in the interior ofthe vesicle, or the interior surface of the wall may be formed byamphiphilic molecules oriented such that their hydrophobic regions arein contact with hydrophobic materials present in the interior of thevesicle.

The amphiphilic molecules in the wall will tend to form layers, andtherefore, the wall may comprise one or more layers of amphiphilicmolecules. If the wall consists of one layer, it may be referred to as a“unilayer membrane” or “monolayer membrane.” If the wall consists of twolayers, it may be referred to as a “bilayer membrane.” Walls with morethan two layers, up to any number of layers, are also within the scopeof the present invention.

The vesicle may be referred to herein as a “buckysome.”

“C_(n)” refers to a fullerene moiety comprising n carbon atoms.Buckminsterfullerenes, also known as fullerenes or, more colloquially,buckyballs, are cage-like molecules consisting essentially ofsp²-hybridized carbons. Fullerenes are the third form of pure carbon, inaddition to diamond and graphite. Typically, fullerenes are arranged inhexagons, pentagons, or both. Most known fullerenes have 12 pentagonsand varying numbers of hexagons depending on the size of the molecule.Common fullerenes include C₆₀ and C₇₀, although fullerenes comprising upto about 400 carbon atoms are also known. Herein,

is used as a representation of a C₆₀ molecule or a C₆₀ moiety in amolecule.

Fullerenes can be produced by any known technique, including, but notlimited to, high temperature vaporization of graphite. Fullerenes are orare expected to be commercially available from MER Corporation (Tucson,Ariz.) and Frontier Carbon Corporation, among other sources.

The naming of specific substituted C₆₀ isomers is complex. Within thepresent specification, the so-called Hirsch Scheme (Hirsch, Angew. Chem.Intl. Ed. (1994) 33(4):437-438) will be used.

Methods of substituting fullerenes with various substituents are wellknown in the art. Methods include 1,3-dipolar additions (Sijbesma etal., J. Am. Chem. Soc. (1993) 115:6510-6512; Suzuki, J. Am. Chem. Soc.(1992) 114:7301-7302; Suzuki et al., Science (1991) 254:1186-1188; Pratoet al., J. Org. Chem. (1993) 58:5578-5580; Vasella et al., Angew. Chem.Int. Ed. Engl. (1992) 31:1388-1390;Pratoetal., J. Am. Chem. Soc. (1993)115:1148-1150; Maggini et al., Tetrahedron Lett. (1994) 35:2985-2988;Maggini et al., J. Am. Chem. Soc. (1993) 115:9798-9799; and Meier etal., J. Am. Chem. Soc. (1994) 116:7044-7048), Diels-Alder reactions(Iyoda et al., J. Chem. Soc. Chem. Commun. (1994) 1929-1930; Belik etal., Angew. Chem. Int. Ed. Engl. (1993) 32:78-80; Bidell et al., J.Chem. Soc. Chem. Commun. (1994) 1641-1642; and Meidine et al., J. Chem.Soc. Chem. Commun. (1993) 1342-1344), other cycloaddition processes(Saunders et al., Tetrahedron Lett. (1994) 35:3869-3872; Tadeshita etal., J. Chem. Soc. Perkin. Trans. (1994) 1433-1437; Beer et al., Angew.Chem. Int. Ed. Engl. (1994) 33:1087-1088; Kusukawa et al.,Organometallics (1994) 13:4186-4188; Averdung et al., Chem. Ber. (1994)127:787-789; Akasaka et al., J. Am. Chem. Soc. (1994) 116:2627-2628; Wuet al., Tetrahedron Lett. (1994) 35:919-922; and Wilson, J. Org. Chem.(1993) 58:6548-6549); cyclopropanation by addition/elimination (Hirschet al., Agnew. Chem. Int. Ed. Engl. (1994) 33:437-438 and Bestmann etal., C. Tetra. Lett. (1994) 35:9017-9020); and addition ofcarbanions/alkyl lithiums/Grignard reagents (Nagashima et al., J. Org.Chem. (1994) 59:1246-1248; Fagan et al., J. Am. Chem. Soc. (1994)114:9697-9699; Hirsch et al., Agnew. Chem. Int. Ed. Engl. (1992)31:766-768; and Komatsu et al., J. Org. Chem. (1994) 59:6101-6102);among others.

The synthesis of substituted fullerenes is reviewed by Murphy et al.,U.S. Pat. No. 6,162,926.

It has been found that fullerenes, especially C₆₀, readily receive up tosix adducts in an octahedral addition pattern (an octahedron having sixvertices) (Brettreich et al., Angew. Chem. Int. Ed. (2000)39:1845-1848).

B is chosen from any organic moiety comprising from 1 to about 40 polarheadgroup moieties. A “polar headgroup” is a moiety which is polar, bywhich is meant that the vector sum of the bond dipoles of each bondwithin the moiety is nonzero. A polar headgroup can be positivelycharged, negatively charged, or neutral. The polar headgroup can belocated such that at least a portion of the moiety can be exposed to theenvironment of the molecule. Exemplary polar headgroup moieties caninclude, but are not limited to, carboxylic acid, alcohol, amide, andamine moieties, among others known in the art. Preferably, B has fromabout 6 to about 24 polar headgroup moieties. In one embodiment, B has astructure wherein the majority of the polar headgroup moieties arecarboxylic acid moieties, which are exposed to water when thesubstituted fullerene is dissolved in an aqueous solvent. A dendrimericor other regular highly-branched structure is suitable for the structureof B.

The value of b can be any integer from 1 to 5. In one embodiment, ifmore than one B group is present (i.e., b>1), that all such B groups areadjacent to each other. By “adjacent” in this context is meant that no Bgroup has only A groups, as defined below, and/or no substituent groupsat all the nearest neighboring points of addition. In the case of anoctahedral addition pattern when b>1, “adjacent” means that the fourvertices of the octahedron in closest proximity to the B group are notall A groups or null.

In one embodiment, B comprises 18 polar headgroup moieties and b=1.

The polar headgroup moieties of B tend to make the B group or groupshydrophilic.

Each B is bonded to C_(n) through a covalent bond or bonds. Any covalentbond which a fullerene carbon is capable of forming and will not disruptthe fullerene structure is contemplated. Examples include carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds. One or more atoms, such as oneor two atoms, of the B group can participate in bonding to C_(n). In oneembodiment, one carbon atom of the B group is bonded to two carbon atomsof C_(n), wherein the two carbon atoms of C_(n) are bonded to eachother.

In one embodiment, B has the amide dendron structure>C(C(═O)OC₃H₆C(═O)NHC(C₂H₄C(═O)NHC(C₂H₄C(═O)OH)₃)₃)₂.

In structure I, A is an organic moiety comprising a terminus proximal tothe C_(n) and one or more termini distal to the C_(n). In oneembodiment, the organic moiety comprises two termini distal to C_(n). By“terminus proximal to C_(n)” is meant a portion of the A group thatcomprises one or more atoms, such as one or two atoms, of the A groupwhich form a bond or bonds to C_(n). By “terminus distal to C_(n)” ismeant a portion of the A group that does not comprise any atoms whichform a bond or bonds to C_(n), but that does comprise one or more atomswhich form a bond or bonds to the terminus of the A group proximal toC_(n).

Each terminus distal to the C_(n) comprises —C_(x)H_(y), wherein x is aninteger and 8≦x≦24, and y is an integer and 1≦y≦2x+1. The —C_(x)H_(y)can be linear, branched, cyclic, aromatic, or some combination thereof.Preferably, A comprises two termini distal to C_(n), wherein each—C_(x)H_(y) is linear, 12≦x≦18, and y=2x+1. More preferably, in each ofthe two termini, x=12 and y=25.

The termini distal to C_(n) tend to make the A groups hydrophobic orlipophilic.

The value of a can be any integer from 1 to 5. Preferably, a is 5. Inone embodiment, if more than one A group is present (i.e., a>1), allsuch A groups are adjacent to each other. By “adjacent” in this contextis meant that no A group has only B groups, as defined below, and/or nosubstituent groups at all the nearest neighboring points of addition. Inthe case of an octahedral addition pattern, when a>1, “adjacent” meansthat the four vertices of the octahedron in closest proximity to the Agroup are not all B groups or null.

Each A is bonded to C_(n) through a covalent bond or bonds. Any covalentbond which a fullerene carbon is capable of forming and will not disruptthe fullerene structure is contemplated. Examples include carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds. One or more atoms, such as oneor two atoms, of the A group can participate in bonding to C_(n). In oneembodiment, one carbon atom of the A group is bonded to two carbon atomsof C_(n), wherein the two carbon atoms of C_(n) are bonded to eachother.

In one embodiment, A has the structure >C(C(═O)O(CH₂)₁₁CH₃)₂.

The number of B and A groups is chosen to be from 2 to 6, i.e., 2≦b+a≦6.In one embodiment, b+a=6. The combination of hydrophilic B group(s) andhydrophobic A group(s) renders the substituted fullerene amphiphilic.The number and identity of B groups and A groups can be chosen toproduce a fullerene with particular amphiphilic qualities which may bedesirable for particular intended uses.

In one preferred embodiment, the substituted fullerene has structure II:

wherein X′ is >C(C(═O)OC₃H₆C(═O)NHC(C₂H₄C(═O)NHC(C₂H₄C(═O)OH)₃)₃)₂ andeach X is >C(C(═O)O(CH₂)₁₁CH₃)₂. A structural representation of asubstituted fullerene having structure II is given in FIG. 1, whereineach X is >C(C(═O)O(CH₂)₁₁CH₃)₂.

In one embodiment, the substituted fullerene has the structure shown inFIG. 1.

The substituted fullerene can further comprise one or more functionalgroups covalently linked to one or more B groups, one or more A groups,or both. In one embodiment, the one or more functional groups arecovalently linked to one or more B groups.

By “functional group” is meant a group that binds to a specificcompound, and thus allows the substituted fullerene to be associatedwith the specific compound.

In one embodiment, the functional group is biotin or a biotin-containingmoiety, i.e., a moiety which will bind to streptavidin.

In another embodiment, the functional group is an antigen-bindingmoiety, by which is meant a moiety comprising the antigen-recognitionsite of an antibody. Examples of a moiety comprising theantigen-recognition site of an antibody include, but are not limited to,monoclonal antibodies, polyclonal antibodies, Fab fragments ofmonoclonal antibodies, Fab fragments of polyclonal antibodies, Fab₂fragments of monoclonal antibodies, and Fab₂ fragments of polyclonalantibodies, among others. Single chain or multiple chainantigen-recognition sites can be used. Multiple chainantigen-recognition sites can be fused or unfused.

The antigen-binding moiety can be selected from any known class ofantibodies. Known classes of antibodies include, but are not necessarilylimited to, IgG, IgM, IgA, IgD, and IgE. The various classes also canhave subclasses. For example, known subclasses of the IgG class include,but are not necessarily limited to, IgG1, IgG2, IgG3, and IgG4. Otherclasses have subclasses that are routinely known by one of ordinaryskill in the art.

The antigen-binding moiety can be selected from an antibody derived fromany species. “Derived from,” in this context, can mean either preparedand extracted in vivo from an individual member of a species, orprepared by known biotechnological techniques from a nucleic acidmolecule encoding, in whole or part, an antibody peptide comprisinginvariant regions which are substantially identical to antibodiesprepared in vivo from an individual member of the species or an antibodypeptide recognized by antisera specifically raised against antibodiesfrom the species. Exemplary species include, but are not limited to,human, chimpanzee, baboon, other primate, mouse, rat, goat, sheep, andrabbit, among others known in the art. In one embodiment, the antibodyis chimeric, i.e., comprises a plurality of portions, wherein eachportion is derived from a different species. A chimeric antibody,wherein one of the portions is derived from human, can be considered ahumanized antibody.

Antigen-recognition moieties are available that recognize antigensassociated with a wide variety of cell types, tissues, and organs, and awide variety of medical conditions, in a wide variety of mammalianspecies. Exemplary medical conditions include, but are not limited to,cancers, such as lung cancer, oral cancer, skin cancer, stomach cancer,colon cancer, nervous system cancer, leukemia, breast cancer, cervicalcancer, prostate cancer, and testicular cancer; arthritis; infections,such as bacterial, viral, fingal, or other microbial infections; anddisorders of the skin, the eye, the vascular system, or other celltypes, tissues, or organs; among others.

Exemplary antigen-recognition moieties known in the art include, but arenot limited to, those derived from antibodies against vascularendothelial growth factor receptor (VEGF-r) (available from Imclone, NewYork, N.Y.), antibodies against epidermal growth factor receptor (EGF-r)(available from Abgenix, Fremont, Calif.), antibodies againstpolypeptides associated with lung cancers (available from CorixaCorporation, Seattle, Wash.), antibodies against human tumor necrosisfactor alpha (hTNF-α) (available from BASF A.G., Ludwigshafen, Germany),among others known in the art.

Antigen-recognition moieties can be prepared by various techniques knownin the art. These techniques include, but are not limited to, theimmunological technique described by Kohler and Milstein in Nature 256,495-497 (1975) and Campbell in “Monoclonal Antibody Technology, TheProduction and Characterization of Rodent and Human Hybridomas” inBurdon et al., Eds., Laboratory Techniques in Biochemistry and MolecularBiology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); aswell as by the recombinant DNA techniques described by Huse et al inScience 246, 1275-1281 (1989); among other techniques known to one ofordinary skill in the art.

In a further embodiment, the functional group is a tissue-recognitionmoiety, by which is meant a moiety that recognizes cells of a particulartissue by binding specifically with one or more proteins expressed bycells of the tissue and present on the exterior of the cells. Examplesof such moieties include, but are not limited to, peptides, among otherclasses of moieties. The term “peptides,” as used herein, encompassesany peptide comprising 1 or more amino acids. Exemplary peptidesinclude, but are not limited to, VEGF, EGF, other growth factors, andother ligands for receptors (such as cell surface receptors, cytoplasmicreceptors, and nuclear receptors), among others.

In one embodiment, wherein the polar headgroups are carboxylic acidmoieties and the functional group is a peptide, a functional group canbe linked to a polar headgroup via an amino linkage between a carboxylicacid in the polar headgroup and an amine in the peptide.

Tissue-recognition moieties can be derived from any species or pluralityof species, and can be selected to target any cell type, tissue, ororgan, or treat any disease.

The inclusion of functional groups will enhance targeting of asubstituted fullerene to a particular tissue. The inclusion offunctional groups in at least some of the substituted fullerenemolecules of the vesicle membrane will enhance the targeting of thevesicle to a particular tissue.

The functional group can also comprise a linker or linkers, i.e.,moieties which are covalently bonded to both (a) the biotin-containingmoiety, the antigen-binding moiety, or the tissue-recognition moiety, asdefined above, and (b) the substituted fullerene, as defined above. Inone embodiment, wherein the polar headgroups are carboxylic acidmoieties, the linker can be an ester.

If some of the substituted fullerene molecules in the vesicle membranecomprise a functional group, from about 0.01 mole % to about 100 mole %of the substituted fullerene molecules of the vesicle membrane comprisethe functional group. In the interest of reduced expense, and in lightof the observation that many functional groups are highly sensitive tothe specific compounds which they bind, preferably from about 0.01 mole% to about 1 mole % of the substituted fullerene molecules of thevesicle membrane comprise the functional group.

In one embodiment, the vesicle wall comprises at least about 50 mol % ofthe substituted fullerene. The balance of the vesicle membrane comprisesother amphiphilic compounds. By “amphiphilic compound” in this contextis meant a compound whose molecules each comprise hydrophobic andhydrophilic regions. Such amphiphilic compounds include, but are notlimited to, commercially-available lipids, such as dimethyl dioctadecylammonium bromide, phosphatidylcholine, and dioleoyltrimethylammoniumphosphate, among others.

In one embodiment, the vesicle wall comprises at least about 75 mol % asubstituted fullerene having structure I. In another embodiment, thevesicle wall consists essentially of a substituted fullerene havingstructure I.

In one embodiment, the vesicle wall is a bilayer membrane. The bilayermembrane comprises two layers, an interior layer formed from substitutedfullerene and other amphiphilic compound or compounds, if any, whereinsubstantially all substituted fullerene and other amphiphilic moleculesare oriented with their hydrophobic portions toward the exterior layer,and an exterior layer formed from substituted fullerene and otheramphiphilic compound or compounds, if any, wherein substantially allsubstituted fullerene and other amphiphilic molecules are oriented withtheir hydrophobic portions toward the interior layer. As a result, thehydrophilic portions of substantially all molecules of each of theinterior and exterior layers are oriented towards aqueous solvent in thevesicle interior or exterior to the vesicle.

Because the hydrophilicity of the hydrophilic portions of the moleculesmay change if the pH or other parameters of the solvent are changed(e.g., if the pH is increased above the pKa of the polar headgroupmoieties of the B groups of the substituted fullerenes, the substitutedfullerenes will readily separate from the vesicle membrane and enter theaqueous phase), the pH and other parameters of the solvent can beadjusted as a matter of routine experimentation by one of ordinary skillin the art in order to allow vesicle formation.

Because the vesicle comprises an interior, and the interior comprises anaqueous solvent, the vesicle can further comprise a therapeutic agent inthe interior of the vesicle. Typically, such a compound is introduced tothe interior of the vesicle as part of the process of forming thevesicle, e.g., by introducing the therapeutic agent, the substitutedfullerene, and other amphiphilic compounds, if any, into an aqueoussolvent under pH and other conditions whereby the substituted fullereneand other amphiphilic compounds, if any, self-assemble a vesicle, withmolecules of the therapeutic agent being sequestered in the vesicleduring vesicle self-assembly. To facilitate self-assembly, preferablythe pH of the solvent is less than about 8.0. However, other techniquesof including a therapeutic agent in the interior of the vesicle known inthe art can be used.

In one embodiment, when the interior of the vesicle comprises water andsubstantially does not comprise a hydrophobic solvent, the therapeuticagent is a water-soluble drug or other compound which, uponadministration to a mammal, can alleviate a medical condition from whichthe mammal suffers. In one embodiment, the therapeutic agent is selectedfrom the group consisting of water-soluble anti-cancer drugs.

In one embodiment, the vesicle wall is a monolayer membrane, in whichmolecules of the substituted fullerene and other amphiphiliccompound(s), if any, are substantially all oriented such that theirhydrophilic regions are adjacent to a polar or aqueous phase, either inthe vesicle interior or exterior to the vesicle, and their hydrophobicregions are adjacent to an apolar phase, either in the vesicle interioror exterior to the vesicle. In this context, “polar” and “apolar” arerelative terms, in that a phase with greater hydrophilicity, miscibilitywith water, etc. is more polar than a phase with poorer solubility inwater. In one embodiment, in the vesicle the hydrophobic regions ofsubstantially all the molecules of the monolayer membrane are orientedtoward the interior of the vesicle.

In one embodiment, when the interior of the vesicle comprises ahydrophobic solvent or other apolar material and substantially does notcomprise water, the therapeutic agent is a hydrophobic drug or othercompound which, upon administration to a mammal, can alleviate a medicalcondition from which the mammal suffers. The terms “hydrophobic” and“lipophilic” are synonyms wheresoever they appear herein.

Any hydrophobic compound can be included in the vesicle interior,typically by providing the substituted fullerene, other amphiphiliccompounds, if any, and the hydrophobic compound in an aqueous solventunder pH and other conditions wherein a monolayer membrane will form,and allowing the vesicle to self-assemble, during which process thehydrophobic compound will be sequestered in the interior of the vesicle.To facilitate assembly of the vesicle, preferably the pH of the solventis less than about 8.0.

The vesicle can be unilamellar (having a single bimolecular membrane),multilamellar (having a plurality of bimolecular membranes,“onion-like”) or hemilamellar (having a single unimolecular membrane).The vesicle can have a size from about 50 Angstroms to about 10 microns.The size, number and nature of membranes, and other parameters of thevesicle can be adjusted as a matter of routine experimentation.

In one embodiment, the therapeutic agent is associated with a fullerene,such as a substituted fullerene comprising a functional group, amongothers. The association can be a covalent link between the fullerenecore and the therapeutic agent; a covalent link between a substituent ofthe fullerene, if any, and the therapeutic agent; an ionic associationbetween a positively- or negatively-charged group on the substituent ofthe fullerene and an oppositely-charged group on the therapeutic agent;or the encapsulation of the therapeutic agent in the fullerene core,among others. A covalent link can be direct or it can make use of acovalent linker linking the therapeutic agent and the fullerene core orsubstituent, if any, of the fullerene. In one embodiment, the covalentlink can be cleaved by an appropriate cleaving technique, such asphotolysis, enzymatic cleavage, or chemical cleavage, among others. Inanother embodiment, a non-covalent association between the therapeuticagent and the fullerene can be dissociated by application of anappropriate chemical, e.g., when the association is an ionicassociation, the association can be dissociated by application of acharged compound of the same charge as the charged group on thetherapeutic agent. Other techniques for dissociating a non-covalentassociation between the substituted fullerene and the therapeutic agentwill be apparent to one of ordinary skill in the art in light of thepresent specification. A chemical or enzyme used to promote dissociationcan be referred to as an “adjuvant.”

Any therapeutic agent, from any source, can be used. As is known in theart, therapeutic agents can be invented by rational drug design,combinatorial chemistry, or serendipitous discovery, among other knowntechniques. Naturally occurring therapeutic agents can be derived from aplant, an animal, a bacterium, a fungus, a virus, or another organism.Therapeutic agents can be synthesized by known chemical synthesistechniques.

The therapeutic agent can treat any disease. Exemplary diseases include,but are not limited to, cancers, autoimmune diseases, infections, liverdiseases, and neurological diseases, among many others.

In one embodiment, the therapeutic agent is an anti-cancer drug.Examples of anti-cancer drugs include paclitaxel (commercially availableas Taxol, Bristol-Myers Squibb), doxorubicin (also known under the tradename Adriamycin), vincristine (known under the trade names Oncovin,Vincasar PES, and Vincrex), actinomycin D, altretamine, asparaginase,bleomycin, busulphan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine,daunorubicin, epirubicin, etoposide, fludarabine, fluorouracil,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,melphalan, mercaptopurine, methotrexate, mitomycin, mitozantrone,oxaliplatin, procarbazine, steroids, streptozocin, taxotere,tamozolomide, thioguanine, thiotepa, tomudex, topotecan, treosulfan, UFT(uracil-tegufur), vinblastine, and vindesine, among others.

Other therapeutic agents include, but are not limited to, the following:hydrocodone, atorvastatin, estrogen, atenolol, levothyroxine,azithromycin, furosemide, amoxicillin, amlodipine, alprazolam,albuterol, loratadine, hydrochlorothiazide, omeprazole, sertraline,paroxetine, triamterene, lansoprazole, ibuprofen, celecoxib,simvastatin, cephalexin, metformin, rofecoxib, lisinopril, amoxicillin,clavulanate, propoxyphene, progesterone, prednisone, norgestimate,ethinyl estradiol, acetaminophen, codeine, cetirizine, fexofenadine,levothyroxine, amoxicillin, metoprolol, lorazepam, metoprolol,fluoxetine, ranitidine, zolpidem, citalopram, amitriptyline,alendronate, quinapril, sildenafil citrate,pravastatin, naproxen,gabapentin, warfarin, ciprofloxacin, verapamil, digoxin, albuterol,bupropion, lisinopril, clonazepam, tramadol, cyclobenzaprine, trazodone,fluticasone, montelukast, diazepam, isosorbide mononitrate s.a.,glyburide, venlafaxine, levofloxacin, medroxyprogesterone, amoxicillin,fluconazole, enalapril, warfarin, carisoprodol, trimeth, sulfameth,fluticasone propionate, benazepril, mometasone, doxycycline, estradiol,allopurinol, rosiglitazone maleate, clopidogrel, propranolol,amlodipine, benazepril, methylprednisolone, valsartan, losartan,insulin, clonidine, diltiazem, loratidine, pseudoephedrine, latanoprost,pioglitazone, loratidine, pseudoephedrine, risperidone, fexofenadine,pseudoephedrine, doxazosin, raloxifene, norethindrone, folic acid,penicillin, oxycodone, temazepam, diltiazem, salmeterol, fosinopril,oxycodone, ramipril, promethazine, terazosin, olanzapine, gemfibrozil,levothyroxine, norethindrone, sumatriptan, hydroxyzine, meclizine,losartan, rabeprazole, phenytoin, clarithromycin, glimepiride,pantoprazole, spironolactone, ipratropium, albuterol, tamsulosin,penicillin, lisinopril, metoclopramide, minocycline, bisoprolol,digoxin, valsartan, metronidazole, cefprozil, triamcinolone, glipizide,norethindrone, levonorgestrel, cefuroxime, nystatin, captopril,promethazine, codeine, acyclovir, norgestimate, oxycodone, irbesartan,nefazodone, mirtazapine, valacyclovir, methylphenidate, cerivastatin,fluoxetine, nitrofurantoin, loratadine, glyburide, metformin, metformin,diltiazem, desogestrel, mupirocin, 1-norgestrel, fluvastatin, aspirin,clarithromycin, clindamycin, esomeprazole, metaxalone, nortriptyline,cimetidine, fenofibrate, iprotropium bromide, tamoxifen, calcitoninsalmon, felodipine, levonorgestrel, salmeterol, fluticasone,theophylline, tetracycline, tolterodine, gatifloxacin, nifedipine,diclofenac, triamcinolone acetonide, promethazine, indomethacin,benzonatate, phenobarbital, naproxen sodium, mometasone, hydrocodone,glipizide, divalproex, nitroglycerin, and phenazopyridine, among others.

One or more therapeutic agents can be used in any composition or methodof the present invention.

The mode of action of the therapeutic agent can be chemotherapeutic,radiotherapeutic, or possess another mode of action. In one embodiment,the therapeutic agent can mediate the application of light, heat, orother external energy in a manner which allows the light, heat, or otherexternal energy to perform a therapeutic action.

In one embodiment, the therapeutic agent is present between two layersof the wall. The therapeutic agent, in this embodiment, may be awater-soluble compound or a lipophilic compound.

In another embodiment, the vesicle can comprise a sensor molecule. A“sensor molecule,” as used herein, is a molecule which can selectivelyassociate with a particular atom or molecule. Any sensor molecule knownin the art can be used. The sensor molecule can be linked with afullerene or anchored in the vesicle wall by hydrophobic, hydrophilic,or both types of interactions.

In a further embodiment, the vesicle can comprise a diagnostic agent. A“diagnostic agent,” as used herein, is a molecule which can be readilydetected by the application of electromagnetic radiation, heat, cooling,the measurement of radioactivity, or other techniques known in the art.The diagnostic agent can be linked with a fullerene or anchored in thevesicle wall by hydrophobic, hydrophilic, or both types of interactions.

In another embodiment, the present invention relates to a method ofadministering a therapeutic agent to a mammal, comprising:

(i) administering a solution comprising a pharmaceutically effectiveamount of the therapeutic agent, wherein the therapeutic agent ispresent in (a) the interior of a vesicle having an interior, anexterior, and a wall, (b) a portion of the wall between two layers, or(c) both, to the mammal, wherein the wall comprises one or more layersand each layer comprises a substituted fullerene having structure I.

The vesicle, the substituted fullerene, and the therapeutic agent are asdescribed above. In one embodiment, the vesicle wall comprises at leastabout 75 mol % a substituted fullerene having structure I. In anotherembodiment, the vesicle wall consists essentially of a substitutedfullerene having structure I.

In one embodiment, the vesicle wall is a monolayer membrane. In anotherembodiment, the vesicle wall is a bilayer membrane.

In one embodiment, from about 0.01 mole % to about 100 mole % of thesubstituted fullerene molecules in the vesicle wall further comprise afunctional group covalently linked to a B group and the functional grouprecognizes a tissue. In another embodiment, the functional group isselected from the group consisting of biotin-containing moieties,antigen-binding moieties, and tissue-recognition moieties, as definedabove.

The pharmaceutically effective amount of the therapeutic agent will varydepending on the compound, the intended effect thereof, theadministration regimen, and the body weight or other characteristics ofthe mammal, among others apparent to one of ordinary skill in the art.The dose of the therapeutic agent will typically be in the range of fromabout 0.001 mg/kg body weight to about 1000 mg/kg body weight. In oneembodiment, the dose of the therapeutic agent will typically be withinthe above range and greater than about 0.01 mg/kg body weight. Inanother embodiment, the dose of the therapeutic agent will typically bewithin the above range and less than about 100 mg/kg body weight.

In one embodiment, the therapeutic agent is an anti-cancer drug. Morepreferably, the anti-cancer drug is selected from the group consistingof paclitaxel, doxorubicin, and vincristine.

Any technique for incorporating the therapeutic agent in the vesicleinterior or between layers of the vesicle wall can be used. An exemplarytechnique is described above, wherein the vesicle is formed in thepresence of the therapeutic agent in an aqueous solvent under pH andother conditions wherein the vesicle can form. This technique can beperformed on either a batch or a continuous basis. Other techniques,however, can be used, such as microinjection of a solution of thetherapeutic agent into the vesicle interior, among others.

In the administering step, a solution comprising the vesicle and thetherapeutic agent in the interior thereof, between two layers of thewall thereof, or both is introduced into the mammal. The solutioncomprises a polar or aqueous solvent and the vesicle comprising thetherapeutic agent. The solution can further comprise adjuvants,preservatives, and other compounds whose inclusion in light of theformation, storage, and/or use of the solution may be desirable.

Any mammal for which it is desired to introduce the therapeutic agentcan be the subject of the method. In one embodiment, the mammal is ahuman.

The term “administering,” as used herein, is intended to encompass alltechniques of introducing a compound to a mammal. Exemplary routes ofadministration include transdermal, subcutaneous, intravenous,intraarterial, intramuscular, oral, rectal, and nasal, among others.

In one embodiment, the vesicle comprises substituted fullerenes furthercomprising a functional group with identity and concentration asdescribed above. A vesicle comprising such substituted fullerenes can bedirected toward a particular tissue.

“Particular tissue” in this context is not meant to be limiting to onecell type, but may be meant to refer to specific bodily fluids, specificorgans comprising a variety of tissues, etc. Particular tissues to whichit may be desirable to direct the vesicles include, but are not limitedto, gastrointestinal tissues, circulatory tissues, lymphatic tissues,biliary tissues, cerebrospinal fluid, synovial fluid, the aqueous humorof the eye, and tumors in the foregoing or any other tissue or celltype.

Fullerenes themselves generally have toxicological properties similar tothose of carbon, and substituted fullerenes are generally not expectedto possess toxic activities. For example, repeated transdermaladministration of fullerenes in benzene for up to 24 weeks (dose=200μg/day) to mice did not result in either benign or malignant skin tumorformation (Nelson et al., Toxicology & Indus. Health (1993)9(4):623-630). Further, no effect on either DNA synthesis or omithinedecarboxylase activity in dermal cells was observed over a 72-hr timecourse after treatment. Zakharenko et al., Doklady Akademii Nauk. (1994)335(2):261-262, have shown that C₆₀ did not produce chromosomal damageat relatively high doses.

In one embodiment, the functional group interacts directly with theparticular tissue. For example, if the functional group is anantigen-binding moiety, and the antigen recognized by the moiety is aprotein present on the surface of cells of a particular tissue, then thefunctional group will bind the protein and direct the vesicle to theparticular tissue. In another example, if the functional group is atissue-recognition moiety, then the functional group will recognize aparticular tissue and direct the vesicle to the particular tissue.Antigen-binding and tissue-recognition moieties, and the antigens theybind and tissues they recognize, are well known in the art. Other directinteractions between the functional group and a particular tissue arepossible and are contemplated as part of the present invention.

In another embodiment, the functional group interacts indirectly with aparticular tissue. By this is meant that the functional group interactswith an adjuvant, and the adjuvant interacts with a particular tissue.“Adjuvant” as used herein refers to any molecule, whether occurring invivo or introduced by administration to the mammal, which provides abeneficial function. In one embodiment, the adjuvant comprises anantigen-binding moiety or a tissue-recognition moiety and a streptavidinmoiety, and the functional group of the substituted fullerene comprisesa biotin-containing moiety. The vesicle interacts with the adjuvantthrough the biotin-containing moiety of the substituted fullerene andthe streptavidin moiety of the adjuvant, and the adjuvant interacts witha particular tissue through the antigen-binding moiety ortissue-recognition moiety as described above.

Adjuvants can provide other or additional beneficial functions. In oneembodiment, an adjuvant facilitates union of the vesicle with themembrane of a cell of a tissue. The union of the vesicle with themembrane will lead to introduction of the therapeutic agent containedwithin the vesicle into the cytoplasm of the cell.

Many useful adjuvants are not present in vivo in the mammal. Therefore,in one embodiment, the method further comprises administering anadjuvant to the mammal, wherein the adjuvant facilitates recognition ofthe tissue by a functional group, union of the vesicle with the membraneof a cell of the tissue, or both. The adjuvant is as described above,and can be administered via any route of administration, as describedabove. The adjuvant can be administered before, after, or simultaneouslywith the solution comprising the vesicle. Typically, the adjuvant isadministered via the same route as the solution comprising the vesicle,but the adjuvant can be administered by a different route, if desired.

In one embodiment, the method allows the systemic distribution of thetherapeutic agent to the mammal, through in vivo disaggregation of thevesicle. The method allows the direction of the vesicle comprising thetherapeutic agent to a particular tissue. In many cases, it is desirableto release the therapeutic agent when the vesicle is in close proximityto the particular tissue.

One technique by which the therapeutic agent can be released in closeproximity to the tissue involves union of the vesicle with the membraneof a cell of the tissue. Union can be facilitated by use of particularadjuvants, as described above.

Another set of techniques by which the therapeutic agent can be releasedin close proximity to the tissue involves disaggregation of the vesiclewhen the vesicle is in close proximity to the tissue. One such techniqueinvolves raising the pH of bodily fluids in which the vesicle ispresent. By raising the pH of such bodily fluids, the number of chargedcarboxyl groups (—COO³¹ ) on the B group of the substituted fullerenewill increase and, depending on the pH and the precise structure of thesubstituted fullerene, molecules of the substituted fullerene may findit more favorable in free energy terms to enter the aqueous solutionthan to remain in the vesicle membrane. This leads to disaggregation ofthe vesicle and release of the therapeutic agent. Therefore, in oneembodiment, the method further comprises raising the pH of bodily fluidsin which the solution comprising the vesicle and the therapeutic agentin the interior thereof is present to a pH at which the vesicledisaggregates. In one embodiment, the pH is raised to greater than about11.0.

Any technique appropriate for raising the pH can be used. Typically,raising the pH can be performed by administering a solution to themammal comprising a compound that will raise the pH of bodily fluids,e.g., a basic solution. Such a solution can be administered via anyroute described above or known in the art.

Another technique for disaggregating the vesicle lies in the observationthat some substituted fullerenes, such as some substituted fullerenesformed from Diels-Alder cycloaddition reactions, readily lose theirsubstituent groups at temperatures slightly above room temperaturedepending on the diene structure of the substituent, and that some othersubstituted fullerenes, such as aldehyde-derived adducts, readily losetheir substituent groups under moisture or heat. The B groups, A groups,or both of the substituted fullerene can be chosen to be added to thefullerene through such reactions. As a result, depending on the natureof the substituent groups and other parameters apparent to one ofordinary skill in the art, and as a matter of routine experimentation,the substituted fullerene can be designed such that it loses B groups, Agroups, or both upon or soon after administration. The loss of B groups,A groups, or both would reduce the amphiphilic character of thesubstituted fullerene, and as a result, would reduce its ability to formor maintain the vesicle membrane. This would be expected to disaggregatethe vesicle.

A further technique for disaggregation of the vesicle is naturaldisaggregation when the vesicle is in contact with a bodily fluid. Thisprocess may be accelerated by the presence of factors (proteins, lipids,salts, etc.) which may be present in the bodily fluid. This process mayoccur without further intervention by the operator of the method.

Another technique for disaggregation of the vesicle involves the use ofphotocleavable polar headgroups. Photocleavable moieties, such as—Ar(NO₂)CH₂—, wherein Ar is an aromatic moiety, can be used to link thefullerene core with the polar headgroups, and vesicles can be formedfrom such substituted fullerenes. Upon irradiation of the vesicle ofthis embodiment by electromagnetic radiation of an appropriatewavelength, the photocleavable moiety can be cleaved and the resultingremoval of polar headgroups from the substituted fullerene can lead todisaggregation of the vesicle.

A further technique for disaggregation of the vesicle involves the useof ultrasonic energy. Upon exposing a region of a mammalian body tosufficient ultrasonic energy, vesicles present in the region candisaggregate and release a therapeutic agent, if any, associated withthe vesicle. The vesicles could be present in the region as a result oftargeting to a specific cell type, tissue, or organ, or could be presentin the region as a result of systemic circulation.

Another technique for disaggregation of the vesicle involves the use ofbiological sensor molecules associated with the vesicle, such as sensormoieties covalently linked with a fullerene molecule in the vesicle wallor sensor molecules anchored by hydrophobic, hydrophilic, or both typesof interactions with the vesicle wall. A particular sensor molecule candetect atoms and molecules present in bodily fluids, such as blood.Exemplary atoms and molecules present in blood include glucose;minerals, such as calcium, potassium, or sodium, among others; hormones,such as insulin, thyroid hormone, testosterone, estrogen, or growthfactors, among others; peptides; enzymes; or blood constituents, such asred blood cell surface molecules, white blood cell surface molecules,platelets, or extracellular hemoglobin, among others; among others. Inone embodiment, the sensor molecule can be chosen such that, uponencountering a bodily region wherein the bodily fluid has an excess ofthe atom or molecule detectable by the sensor molecule, the sensormolecule binds the atom or molecule and leaves the vesicle or the sensormolecule undergoes a conformational change. In either case, if thesensor molecule, the vesicle, and other features are properly chosen,the vesicle can disaggregate. Alternatively, the sensor molecule can bechosen such that disaggregation of the vesicle occurs upon encounteringa bodily region wherein the bodily fluid has a shortage of the atom ormolecule.

In one embodiment, the present invention relates to a method ofdiagnosing a medical condition in a mammal, comprising:

(i) administering a solution comprising a pharmaceutically effectiveamount of a diagnostic agent, wherein the diagnostic agent is present in(a) the interior of a vesicle having an interior, an exterior, and awall, (b) a portion of the wall between two layers, or (c) both, whereinthe vesicle is as described above and comprises fullerenes substitutedwith a functional group, as described above, to the mammal; and

(ii) detecting the diagnostic agent.

Any agent which is detectable by any means can be the “diagnosticagent.” In one embodiment, the diagnostic agent is a fluorophore, whichcan be made to fluoresce upon exposure to a particular wavelength ofelectromagnetic radiation. In another embodiment, the diagnostic agentis a radionuclide, which can be detected by known techniques. Otherdiagnostic agents are known in the art.

The method is similar to the method of treatment described above, exceptthat instead of releasing a therapeutic agent to a particular cell typeor tissue, a diagnostic agent is directed to the vicinity of the celltype or tissue by the use of a vesicle comprising fullerenes substitutedwith functional groups such as antigen-binding moieties ortissue-recognition moieties.

In one embodiment, the present invention relates to a method ofadministering a therapeutic agent to a mammal, comprising:

(i) administering a solution comprising (a) a substituted fullerene,comprising (a-i) a fullerene moiety comprising n carbon atoms, wherein nis an integer and 60≦n≦240, and (a-ii) a functional group selected fromthe group consisting of biotin-containing moieties, antigen-bindingmoieties, and tissue-recognition moieties, and (b) a pharmaceuticallyeffective amount of the therapeutic agent, wherein the therapeutic agentis associated with the substituted fullerene.

The substituted fullerene and the therapeutic agent are as describedabove. In one embodiment, the therapeutic agent is an anti-cancer drug.

In one embodiment, the method further comprises (ii) administering anadjuvant to the mammal, wherein the adjuvant facilitates dissociation ofthe therapeutic agent from the substituted fullerene. An appropriateadjuvant for a particular substituted fullerene and a particulartherapeutic agent can be selected in light of the discussion above.

The ability of vesicles, alternatively referred to as liposomes, tofunction as drug delivery vehicles is well known in the art, asdiscussed above and as known from work by Alza Corporation (MountainView, Calif.), the University of California, and Sequus Pharmaceuticals,among other entities.

In another embodiment, the present invention relates to a derivatizedcarbon nanotube, comprising:

a carbon nanotube, and

at least one therapeutic agent,

wherein each therapeutic agent is either covalently attached to thecarbon nanotube or present within the carbon nanotube.

As is well known, carbon has not only the propensity to self-assemblefrom a high temperature vapor to form perfect spheroidal closed cages(fullerenes), but also, with the aid of a transition metal catalyst, toassemble into single-wall cylinders with may be sealed at one or bothends with a semifullerene dome. These tubes may be thought of astwo-dimensional single crystals of carbon. Multi-wall cylinders,comprising nested single-wall cylinders, have also been observed. Bothmulti-wall and single-wall cylinders are encompassed by the term “carbonnanotube,” as used herein.

In defining carbon nanotubes, it is helpful to use a recognized systemof nomenclature. Herein will be used the carbon nanotube nomenclaturedescribed by Dresselhaus et al., Science of Fullerenes and CarbonNanotubes, Ch. 19. Single wall tubular fullerenes are distinguished fromeach other by a double index (n, m), where n and m are integers thatdescribe how to cut a single strip of hexagonal “chicken-wire” graphitesuch that its edges join seamlessly when the strip is wrapped onto thesurface of a cylinder. When n=m, the resultant tube is said to be of the“arm-chair” or (n, n) type, since when the tube is cut perpendicularlyto the tube axis, only the sides of the hexagons are exposed and theirpattern around the periphery of the tube edge resembles the arm and seatof an arm chair repeated n times. Regardless of tube type, allsingle-wall nanotubes have extremely high thermal conductivity andtensile strength. Arm-chair tubes also have extremely high electricalconductivity.

Single-wall carbon nanotubes (SWNTs) are much more likely to be free ofdefects than are multi-wall carbon nanotubes. This is believed to bebecause multi-wall carbon nanotubes can survive occasional defects,whereas SWNTs have no neighboring walls to compensate for defects byforming bridges between unsaturated carbon valences. Since single-wallcarbon nanotubes have fewer defects, they are generally stronger, moreconductive, and typically more useful than multi-wall carbon nanotubesof similar diameter. However, both SWNTs and multi-wall carbon nanotubesmay be used within the scope of the present invention. The precisestructure of an SWNT or a multiwall carbon nanotube is not crucial andis a matter of routine experimentation to one of ordinary skill in theart. SWNTs often have a diameter of about 0.3 nm to about 8 nm. In oneembodiment, the SWNT has a diameter of about 1.2 nm. Multi-wall carbonnanotubes often have a diameter of about 30 nm to about 200 nm.

In one embodiment, the carbon nanotube is a single-wall carbon nanotubewherein m+n=2 to 20. In one embodiment, the carbon nanotube is asingle-wall nanotube with (10,10) structure.

The at least one therapeutic agent is selected from any therapeuticagent which can be covalently bonded to the carbon nanotube. The term“at least one therapeutic agent” encompasses both one or more moleculesof a single therapeutic agent and one or more molecules of each of oneor more therapeutic agent. In one embodiment, the therapeutic agent canbe bonded to the carbon nanotube through one or more bonds between oneor more carbons of the carbon nanotube and one or more atoms of thetherapeutic agent. In one embodiment, the one or more atoms of thetherapeutic agent are selected from the group consisting of carbon,oxygen, and nitrogen. Preferably, the bonds are single bonds.

In this embodiment, any desirable therapeutic agent can be bonded to thecarbon nanotube. In one embodiment, the therapeutic agent is asdescribed above.

In another embodiment, the therapeutic agent can be present within thecarbon nanotube. In one embodiment, the therapeutic agent is asdescribed above.

The derivatized carbon nanotube can further comprise other moieties. Inone embodiment, the derivatized carbon nanotube further comprises afunctional group selected from the group consisting of biotin,biotin-containing moieties, antigen-binding moieties, andtissue-recognition moieties, as described above.

In another embodiment, the present invention relates to a method ofdelivering a therapeutic agent to a mammal, comprising

(i) administering to the mammal a derivatized carbon nanotube,comprising a carbon nanotube and at least one therapeutic agent, whereineach therapeutic agent is covalently attached to the carbon nanotube.

The derivatized carbon nanotube is as described above. In oneembodiment, the derivatized carbon nanotube further comprises afunctional group selected from the group consisting of biotin,biotin-containing moieties, antigen-binding moieties, andtissue-recognition moieties. Such a functional group will enhance thedirection of the derivatized carbon nanotube to a desired tissue of themammal.

The first administering step can be performed via any appropriate routeas described above. Typically, the derivatized carbon nanotube is in anaqueous solution, and the solution can further comprise preservatives,adjuvants, and other compounds known in the art. However, other vehiclesfor the derivatized carbon nanotube (e.g., apolar solution, solid, etc.)can be used.

In one embodiment, the method further comprises:

(ii) administering to the mammal an adjuvant which promotes disruptionof the covalent bond between the carbon nanotube and the at least onetherapeutic agent, thereby delivering the at least one therapeutic agentto the tissue.

The second administering step can be performed via any appropriate routeas described above. The adjuvant administered in the secondadministering step can be any compound that promotes disruption of thecovalent bond linking the at least one therapeutic agent to the carbonnanotube, or can be any compound that enhances the action of thetherapeutic agent in any other way. The adjuvant is typically in anaqueous solution, and the solution can further comprise preservativesand other compounds known in the art.

One embodiment of this method is shown in FIG. 8.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES

The following examples show that the amphiphilic lipofullerene 1(Structure III) is able to form stable liposomes (Example 1). The sizeof the liposomes can be influenced by controlling the pH value of thesolution (Example 2). Furthermore, it is possible to functionalize theacid-units of amphiphilic lipofullerenes with a fluorescence-marker oran anchor molecule (Example 3).

Experimental Techniques

Cryo-TEM

A droplet (5 μl) of freshly prepared amphifullerene solution (0.2%(w/v)) in sodium-phosphate buffer (pH=6.84) was placed on ahydrophilized holey carbon filmed grid, exposed to 60 s plasma treatmentat 8 W using a BALTEC MED 020 device, and excess fluid was blotted offto create an ultrathin layer of the solution spanning the holes of thecarbon film. The grids were immediately vitrified in liquid ethane atits freezing point (89K) using a standard plunging device. The vitrifiedsamples were transferred under liquid nitrogen into a Philips CM12transmission electron microscope using the Gatan cryoholder and -stage(Model 626). Microscopy was carried out at −175° C. sample temperatureusing the microscopes low dose protocol to avoid unnecessaryirradiation. The primary magnification was 58,300× and the defocus waschosen to be 0.9 μm, corresponding to a first zero of the CTF (contrasttransfer function) at 18 Å (Cs=2 mm).

Dynamic Light Scattering

The amphifullerene was dissolved in dust-free Milli-Q-water and filteredonce with either a Millex-GS-Filter (Millipore, pore-size 22 μm) or aMillex-HA-filter (Millipore, pore-size 0.45 μm). All cuvettes and flaskswere made dust-free in an acetone fountain. Dynamic lightscatteringmeasurements were carried out with the following apparatus: Stabilite2060-KR-R krypton ion laser (λ=647.1 nm) from Spectra-Physics,goniometer SP-86 from ALV, and digital correlator ALV-3000 from ALV.

Monolayer-Experiments

The amphifullerene was dissolved in chloroform at a concentration of 0.2nmol/μL. For all monolayer experiments at the air/water interface, thesubphase contained 0.25 mM EDTA (ethylenediamine tetra-acetic acid) andwas buffered with either 25 mM phosphate or 20 mM HEPES(N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid). If not indicatedotherwise, the pH was adjusted by NaOH (approximately 13 mM for thephosphate buffer, approximately 4 mM for the HEPES buffer) to pH 7.0.Buffers were prepared using either Millipore purified water. Monolayersof the amphiphilic fullerene-derivative were spread from the organicsolution using a microsyringe and afterward compressed to the desiredsurface pressure.

Film balance experiments at different pH values were carried out on aLangmuir trough with a maximum surface area of 422 cm² and a subphasetemperature of 20.0±0.2° C. for all experiments. The pK-value of themonolayer was determined employing a dedicated film balance equippedwith an inner “titration compartment” as follows: A monolayer of theamphifullerene was first compressed to 4.5 mN/m on a subphase of pH 4.0.After this, the channel link between the trough and the titrationcompartment was pressure tightly closed and the pH of the compartmentsubphase was successively increased by injecting a total of 500 μL of1.00 M NaOH into the subphase through the injection hole and the changeof π was recorded after appropriate equilibration.

Example 1 Formation of a Variety of Stable aggregates Based on theAmphiphilic Lipofullerene 1 (FIG. 1)

The amphiphilic lipofullerene 1 has the structure shown in FIG. 1. Theamphiphilic lipofullerene (“amphifullerene”) 1 was observed tospontaneously aggregate in aqueous solution at pH 7.4. The aggregateswere stable at least during several days and they did not disaggregatedown to a cmc (critical micelle-building constant) of at least 4×10⁻⁷mol/L.

With light scattering methods and electron microscopy, the size and formof the liposomes were determined. The amphiphilic lipofullerene 1 tendedto form unilamellar (bilayer) vesicles and cylindrical micelles. Thediameter of these vesicles varied from 50 nm to about 400 nm. Thecylindrical micelles had a thickness of 7 nm, which is roughlyequivalent to the size of two molecules, and the cylindrical micellesshowed different lengths varying from 50 to 200 nm. A cryo-TEM of atypical vesicle is shown in FIG. 2. This vesicle had a diameter of about80 nm and a thickness of the bilayer of about 7 nm. The dark regions inthe bilayer represent the C₆₀-core of the amphifullerene.

Example 2 Adjusting the Size of Liposomes by Variation of the pH Value

Due to the pK_(a) values of the amphifullerene 1, a variation of thedegree of protonation is possible and has been observed in a pH rangefrom 6 to 11. Changes in the pH drastically influence the charge densityon the surface of liposomes comprising the amphifullerene. Therefore, itwas considered possible to change the aggregation properties bydiffering the pH. This behavior was demonstrated by pH titrationexperiments on a monolayer of the amphifullerene and by the dependenceof light scattering measurements on pH.

Monolayer Experiments

Even if pH titration of the monolayer does not provide immediateinformation about the aggregation-behavior in solution, informationabout the electrostatic interaction at the vesicle surface is readilyavailable. The pressure at the monolayer is directly related to the pHvalue of the solution and also to the electrostatic interaction betweenthe acid units. With increasing pH, the surface charge increases andwith this increase the propensity to form aggregates would be expectedto decrease. The dependence of pH on the surface pressure is given inFIG. 3.

Light Scattering Experiments in Solution

The pH-dependent altering of the vesicle size in a solution of liposomeswas shown with light-scattering experiments. Table 1 shows that anincrease of pH from about 7 to about 11 led to a reduction in thehydrodynamic radius of the vesicle from about 50 nm to about 19 nm. Weexpect the use of other amphiphilic lipofullerenes would lead to similaradjustments of these values. TABLE 1 Dependence of vesicle size on pH.(D_(app): apparent diffusion coefficient). Rh pH concentration [g/L]D_(ap) _(p)(q² → 0) (hydrodynamic radius) [nm] 7.2 0.6 4.30 × 10⁻⁸ 49.78.4 0.6 4.87 × 10⁻⁸ 43.9 11 0.6 1.14 × 10⁻⁷ 18.7

Example 3 Functionalization of Amphifullerene 1 with a FluorescentMolecule or an Anchor Molecule

In order to attach biomolecules to amphifullerene 1, we used an anchormolecule. The fluorescence marker Texas Red® (sulforhodamine,commercially available from Molecular Probes, Inc., Eugene, Oreg.) wasused in this study.

In another experiment, biotin, which is able to bind to biomolecules(avidin, streptavidin), was attached to amphifullerene 1.

Attachment of Texas Red®

Texas Red® is a fluorophore which is derived from rhodamine and emits ata longer wavelength than other rhodamine derivatives. The preconditionsfor the labeling were set to 1-2% (maximum of 5%) of labeledamphifullerenes and only one fluorophore moiety per dendrimer as far aspossible. The following statistical labeling of the carboxylic endgroups was performed in absolute chloroform with freshly preparedstandard solutions of carbonyldiimidazole (CDI) in absolute chloroformand of Texas Red® in N,N-dimethylformamide (DMF). The amphifullerene 1was first partially activated at the carboxylic acid groups by CDI.After 1 hour, 5 mol % of the amino derivative of the fluorophore wasadded. The coupling was followed by thin layer chromatography (TLC). Thesolution was stirred for 1 day and subsequently diluted with chloroformand washed with water. The orange-colored chloroform phase was thentransferred to a TLC plate and separated by preparative TLC (on a silicaTLC plate). After repeated solvation of the amphifullerene-containingfraction with ethanol and precipitation of dissolved silica withmethylenechloride, the solution was rotary evaporated and the productwas dried in vacuo.

FIG. 7 shows the synthesis of a dendrofullerene hexakisadduct labeledwith the fluorescence marker Texas Red®.

The total yield was 78%. Use of a TLC control verified the purity of theproduct. The ratio of 1 and 2 in the mixture was determined by UV/V isspectroscopy. The strong absorption band of Texas Red® at 589 nmrendered molecules 2 capable of detection and calibration for the smallproportion of fluorophore units (FIG. 4). In literature, an extinctioncoefficient for Texas Red® in DMF of 81×10³ at 591 nm has been reported.Because 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran(DCM) was used for the characterization of 2, the extinction coefficientfor Texas Red® in DCM (presolvated in a small amount of DMF) wasdetermined. The value of 62(±0.3)×10³ was obtained.

The extinction coefficients of the amphifullerene 1 in DCM weredetermined. The values of ε=77×10³ at 271 nm, 79×10³ at 282 nm, 52×10³at 318 nm and 41×10³ at 334 nm are in good agreement with the reportedvalues.

The ratio of the extinction coefficients of amphifullerene 1 andfluorophore Texas Red® was used to determine the amount of labeledmolecules. With an appropriate baseline correction in the fluorophoreregion of the UV/V is spectrum shown in FIG. 5, a labeling ratio of 2.0%was determined.

Attachment of Biotin

In a different attempt to functionalize the amphifullerene, the anchormolecule biotin, which is able to bind to biomolecules (avidin,streptavidin), was attached. Based on the coupling-experiments with thefluorophore a biotin-spacer-molecule was attached to the amphifullerene1 to give the anchor-functionalized amphifullerene 3 (see FIG. 6).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1-34. (canceled)
 35. A substituted fullerene, comprising: a fullerenemoiety comprising n carbon atoms, wherein n is an integer and 60≦n≦240,and a functional group selected from the group consisting ofbiotin-containing moieties, antigen-binding moieties, andtissue-recognition moieties.
 36. The substituted fullerene of claim 35,comprising structure I:(B)_(b)—C_(n)-(A)_(a)   (I) wherein C_(n) is a fullerene moietycomprising n carbon atoms, wherein n is an integer and 60≦n≦240; B is anorganic moiety comprising from 1 to about 40 polar headgroup moieties; bis an integer and 1≦b≦5; each B is covalently bonded to the C_(n)through 1 or 2 carbon-carbon, carbon-oxygen, or carbon-nitrogen bonds; Ais an organic moiety comprising a terminus proximal to the C_(n) and oneor more termini distal to the C_(n), wherein the termini distal to theC_(n) each comprise —C_(x)H_(y), wherein x is an integer and8≦x≦24, andy is an integer and 1≦y≦2x+1; a is an integer, 1≦a≦5; 2≦b+a≦6; and eachA is covalently bonded to the C_(n) through 1 or 2 carbon-carbon,carbon-oxygen, or carbon-nitrogen bonds, wherein the functional group iscovalently linked to a B group.
 37. The substituted fullerene of claim36, wherein B comprises 18 polar headgroup moieties; A comprises twotermini distal to the C_(n); x=12; y=25; b=1; and a=5.
 38. Thesubstituted fullerene of claim 37, comprising structure II:

wherein X′ is >C(C(═O)OC₃H₆C(═O)NHC(C₂H₄C(═O)NHC(C₂H₄C(═O)OH)₃)₃)₂ or afunctional-group substituted>C(C(═O)OC₃H₆C(═O)NHC(C₂H₄C(═O)NHC(C₂H₄C(═O)OH)₃)₃)₂, and each X is>C(C(═O)O(CH₂)₁₁CH₃)₂.
 39. The substituted fullerene of claim 35,further comprising a therapeutic moiety associated with the fullerenemoiety.
 40. The substituted fullerene of claim 39, wherein thetherapeutic moiety is an anti-cancer drug.
 41. A method of administeringa therapeutic agent to a mammal, comprising: (i) administering asolution comprising (a) a substituted fullerene comprising (a-i) afullerene moiety comprising n carbon atoms, wherein n is an integer and60≦n≦240, and (a-ii) a functional group selected from the groupconsisting of biotin-containing moieties, antigen-binding moieties, andtissue-recognition moieties, and (b) a pharmaceutically effective amountof the therapeutic agent, wherein the therapeutic agent is associatedwith the substituted fullerene.
 42. The method of claim 41, wherein thetherapeutic agent is an anti-cancer drug.
 43. The method of claim 41,further comprising administering an adjuvant to the mammal, wherein theadjuvant facilitates dissociation of the therapeutic agent from thesubstituted fullerene.
 44. (canceled)